HIV-associated nephropathy (HIVAN) is the third leading cause of the end-stage renal disease in African Americans between ages 20-64. One of the pathologic features of HIVAN is collapsing focal segmental glomerulosclerosis (FSGS). Glomerular podocytes, the key cells involved in FSGS, exhibit a disease phenotype characterized by proliferation and dedifferentiation in both human and mouse kidneys with HIVAN. We reported that HIV-1 mRNA were expressed in podocytes of HIVAN patients and of HIV-1 transgenic mice. Studies from transgenic mice suggest that Nef, one of the HIV accessory proteins, plays an important role in the pathogenesis of HIVAN. We found that Nef induces podocyte proliferation and loss of contact inhibition in vitro. In addition, we found that Nef induces podocyte dedifferentiation and the rearrangement of actin filaments. Our preliminary data also suggest that Nef activates Src-Stat3, Ras-MAPK, and Rac1/CDC42 pathways in kidney podocytes. In this study, we propose the following specific aims: 1. To determine in podocytes: a) how Nef induces cell proliferation and loss of contact inhibition by examining the effects of Nef on expressions of cyclins, cyclin inhibitors, integrins, and adhesion molecules; b) how Nef affects the expression of differentiation markers, the organization of actin filaments and microtubules, and the arrangement of actin-associated molecules such as synaptopodin, Ezrin, and CD2AP. 2. To determine in podocytes: a) what are the potential Nef-interactive proteins as well as their down-stream signaling pathways; b) to define the relationships between Nef-induced signaling pathways and Nef-induced phenotypic changes using dominant negative mutants for Src, Stat3, Ras, Rac1/CDC42 or mutants for different Nef binding motifs. 3. To verify that these in vitro findings have in vivo relevance by determining: a) whether Nef induces similar signaling pathways and phenotypic changes in primary cultures of murine podocytes; b) whether these phenotypic changes and signaling pathway activations are observed in kidney podocytes of HIV transgenic mice and HIV patients with HIVAN in vivo. These studies should elucidate essential mechanism responsible for the development of HIV-associated nephropathy and how Nef plays a role in this pathologic process.